Bottom Line:
In particular, AtPIP1;4 mediates CO2 transport with a substantial contribute to photosynthesis and further increases this function upon interacting with Hpa1 at the plasma membrane.As a result, leaf photosynthesis rates are increased and the plant growth is enhanced in contrast to the normal process without Hpa1-AtPIP1;4 interaction.Our findings demonstrate the first case that plant sensing of a bacterial harpin protein is connected with photosynthetic physiology to regulate plant growth.

ABSTRACTHarpin proteins produced by plant-pathogenic Gram-negative bacteria are the venerable player in regulating bacterial virulence and inducing plant growth and defenses. A major gap in these effects is plant sensing linked to cellular responses, and plant sensor for harpin Hpa1 from rice bacterial blight pathogen points to plasma membrane intrinsic protein (PIP). Here we show that Arabidopsis AtPIP1;4 is a plasma membrane sensor of Hpa1 and plays a dual role in plasma membrane permeability of CO2 and H2O. In particular, AtPIP1;4 mediates CO2 transport with a substantial contribute to photosynthesis and further increases this function upon interacting with Hpa1 at the plasma membrane. As a result, leaf photosynthesis rates are increased and the plant growth is enhanced in contrast to the normal process without Hpa1-AtPIP1;4 interaction. Our findings demonstrate the first case that plant sensing of a bacterial harpin protein is connected with photosynthetic physiology to regulate plant growth.

f1: Hpa1 requires its N-terminus to interact with AtPIP1;4 in yeast, in vitro, and at Arabidopsis PMs.(a) The split-ubiquitin-based Y2H assay. Three types of synthetic dropout (SD)-amino acid nutrient media were used in screening of yeast hybrids. The SD-WL medium allows growth of yeast cells irrespectively of protein interactions. Yeast cells are able to grow on both SD-WLH and SD-WLAH media only when an interaction of tested proteins occurs. The interaction can be also detected by the X-Gal assay of colonies grown on SD-WL. (b) Immunoblotting of the three proteins analyzed directly (control) and proteins eluted from a glutathione-affinity resin (pulldown), showing that GST-His-Hpa1, but not GST-His-Hpa1∆NT, was able to bind with AtPIP1;4-His in the resin. (c–e) YFP BiFC imaging of (c) protoplasts or (d) and (e) leaves. Scale bars = 10 μm. (c,b) Red-fluorescent PM marker FM 4–64 was used to show cell outlines. (e) To better visualize BiFC signal, the guard cell was focused on the bulgy opening side.

Mentions:
We looked for Hpa1-interacting proteins in Arabidopsis by yeast two-hybrid (Y2H) systems. As a first step, a cDNA prey library from the Arabidopsis ecotype Col-0 was screened with the bait vector containing Hpa1 or Hpa1∆NT. Screening of yeast transformants identified seven Hpa1-interacting clones; five of them also interacted with Hpa1∆NT (Supplementary Fig. 1). The clone containing a partial sequence fragment of the AtPIP1;4 cDNA was further studied as AtPIP1;4 was a candidate that might interact with Hpa1 at the PM. The full-length coding sequence of AtPIP1;4 was isolated from Col-0 and retested by Y2H in crosswise combinations with Hpa1 or Hpa1∆NT as mutual bait and preys. This crosswise assay indicated AtPIP1;4 interaction with both Hpa1 and Hpa1∆NT (Supplementary Fig. 2). Proteins were further tested in a split-ubiquitin-based (SUB) Y2H system. An interaction was observed between AtPIP1;4 and Hpa1, but not between AtPIP1;4 and Hpa1∆NT (Fig. 1a; Supplementary Fig. 3). Then, Hpa1 and Hpa1∆NT were fused to histidine (His) and glutatione S-transferase (GST) tags15, and fusion proteins were analyzed by the in vitro pulldown assay. This assay detected AtPIP1;4 interaction with Hpa1 but not with Hpa1∆NT (Fig. 1b).

f1: Hpa1 requires its N-terminus to interact with AtPIP1;4 in yeast, in vitro, and at Arabidopsis PMs.(a) The split-ubiquitin-based Y2H assay. Three types of synthetic dropout (SD)-amino acid nutrient media were used in screening of yeast hybrids. The SD-WL medium allows growth of yeast cells irrespectively of protein interactions. Yeast cells are able to grow on both SD-WLH and SD-WLAH media only when an interaction of tested proteins occurs. The interaction can be also detected by the X-Gal assay of colonies grown on SD-WL. (b) Immunoblotting of the three proteins analyzed directly (control) and proteins eluted from a glutathione-affinity resin (pulldown), showing that GST-His-Hpa1, but not GST-His-Hpa1∆NT, was able to bind with AtPIP1;4-His in the resin. (c–e) YFP BiFC imaging of (c) protoplasts or (d) and (e) leaves. Scale bars = 10 μm. (c,b) Red-fluorescent PM marker FM 4–64 was used to show cell outlines. (e) To better visualize BiFC signal, the guard cell was focused on the bulgy opening side.

Mentions:
We looked for Hpa1-interacting proteins in Arabidopsis by yeast two-hybrid (Y2H) systems. As a first step, a cDNA prey library from the Arabidopsis ecotype Col-0 was screened with the bait vector containing Hpa1 or Hpa1∆NT. Screening of yeast transformants identified seven Hpa1-interacting clones; five of them also interacted with Hpa1∆NT (Supplementary Fig. 1). The clone containing a partial sequence fragment of the AtPIP1;4 cDNA was further studied as AtPIP1;4 was a candidate that might interact with Hpa1 at the PM. The full-length coding sequence of AtPIP1;4 was isolated from Col-0 and retested by Y2H in crosswise combinations with Hpa1 or Hpa1∆NT as mutual bait and preys. This crosswise assay indicated AtPIP1;4 interaction with both Hpa1 and Hpa1∆NT (Supplementary Fig. 2). Proteins were further tested in a split-ubiquitin-based (SUB) Y2H system. An interaction was observed between AtPIP1;4 and Hpa1, but not between AtPIP1;4 and Hpa1∆NT (Fig. 1a; Supplementary Fig. 3). Then, Hpa1 and Hpa1∆NT were fused to histidine (His) and glutatione S-transferase (GST) tags15, and fusion proteins were analyzed by the in vitro pulldown assay. This assay detected AtPIP1;4 interaction with Hpa1 but not with Hpa1∆NT (Fig. 1b).

Bottom Line:
In particular, AtPIP1;4 mediates CO2 transport with a substantial contribute to photosynthesis and further increases this function upon interacting with Hpa1 at the plasma membrane.As a result, leaf photosynthesis rates are increased and the plant growth is enhanced in contrast to the normal process without Hpa1-AtPIP1;4 interaction.Our findings demonstrate the first case that plant sensing of a bacterial harpin protein is connected with photosynthetic physiology to regulate plant growth.

ABSTRACTHarpin proteins produced by plant-pathogenic Gram-negative bacteria are the venerable player in regulating bacterial virulence and inducing plant growth and defenses. A major gap in these effects is plant sensing linked to cellular responses, and plant sensor for harpin Hpa1 from rice bacterial blight pathogen points to plasma membrane intrinsic protein (PIP). Here we show that Arabidopsis AtPIP1;4 is a plasma membrane sensor of Hpa1 and plays a dual role in plasma membrane permeability of CO2 and H2O. In particular, AtPIP1;4 mediates CO2 transport with a substantial contribute to photosynthesis and further increases this function upon interacting with Hpa1 at the plasma membrane. As a result, leaf photosynthesis rates are increased and the plant growth is enhanced in contrast to the normal process without Hpa1-AtPIP1;4 interaction. Our findings demonstrate the first case that plant sensing of a bacterial harpin protein is connected with photosynthetic physiology to regulate plant growth.